The present invention is related to wound dressing materials and tissue engineering scaffolds. More specifically, the present invention is related to a cross-linked keratin hydrogel.
Chronic wounds can be caused by a variety of events, including surgery, prolonged bedrest and traumatic injuries. Partial thickness wounds can include second degree burns, abrasions, and skin graft donor sites. Healing of these wounds can be problematic, especially in cases of diabetes mellitus or chronic immune disorders. Full thickness wounds have no skin remaining, and can be the result of trauma, diabetes (e.g., leg ulcers) and venous stasis disease, which can cause full thickness ulcers of the lower extremities. Full thickness wounds tend to heal very slowly. Proper wound care technique including the use of wound dressings is extremely important to successful chronic wound management. Chronic wounds affect an estimated four million people a year, resulting in health care costs in the billions of dollars. xe2x80x9cTreatment of Skin Ulcers with Cultivated Epidermal Allografts,xe2x80x9d T. Phillips, O. Kehinde, and H. Green, J. Am. Acad. Dermatol., V. 21, pp. 191-199 (1989).
The wound healing process involves a complex series of biological interactions at the cellular level which can be grouped into three phases: hemostasis and inflammation; granulation tissue formation and reepithelization; and remodeling. xe2x80x9cCutaneous Tissue Repair: Basic Biological Considerations,xe2x80x9d R. A. F. Clark, J. Am. Acad. Dermatol., Vol. 13, pp. 701-725 (1985). Keratinocytes (epidermal cells that manufacture and contain keratin) migrate from wound edges to cover the wound. Growth factors such as transforming growth factor-xcex2 (TGF-xcex2) play a critical role in stimulating the migration process. The migration occurs optimally under the cover of a moist layer. Keratins have been found to be necessary for reepithelization. Specifically, keratin types K5 and K14 have been found in the lower, generating, epidermal cells, and types K1 and K10 have been found in the upper, differentiated cells. Wound Healing: Biochemical and Clinical Aspects, I. K. Cohen, R. F. Diegleman, and W. J. Lindblad, eds., W. W. Saunders Company, 1992. Keratin types K6 and K10 are believed to be present in healing wounds, but not in normal skin. Keratins are major structural proteins of all epithelial cell types and appear to play a major role in wound healing.
An optimum wound dressing would protect the injured tissue, maintain a moist environment, be water permeable, maintain microbial control, deliver healing agents to the wound site, be easy to apply, not require frequent changes and be non-toxic and non-antigenic. Although not ideal for chronic wounds, several wound dressings are currently on the market, including occlusive dressings, non-adherent dressings, absorbent dressings, and dressings in the form of sheets, foams, powders and gels. Wound Management and Dressing, S. Thomas, The Pharmaceutical Press, London, 1990.
Attempts have been made to provide improved dressings that would assist in the wound healing process using biological materials such as growth factors. To date, these biologicals have proven very costly and shown minimal clinical relevance in accelerating the chronic wound healing process. In cases of severe full thickness wounds, autografts (skin grafts from the patient""s body) are often used. Although the graft is non-antigenic, it must be harvested from a donor site on the patient""s body, creating an additional wound. In addition, availability of autologous tissue may not be adequate. Allografts (skin grafts from donors other than the patient) are also used when donor sites are not an option. Allografts essentially provide a xe2x80x9cwound dressingxe2x80x9d that provides a moist, water permeable layer, but is rejected by the patient usually within two weeks and does not become part of the new epidermis.
What would be desirable and has not heretofore been provided is a wound dressing that protects the injured tissue, maintains a moist environment, is water permeable, is easy to apply, does not require frequent changes and is non-toxic and non-antigenic, and most important, delivers effective healing agents to the wound site.
Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The consistent success of a tissue engineered implant rests on the invention of a biocompatible, mitogenic material that can successfully support cell growth and differentiation and integrate into existing tissue. Such a scaffolding material could greatly advance the state of the tissue engineering technologies and result in a wide array of tissue engineered implants containing cellular components such as osteoblasts, chondrocytes, keratinocytes, and hepatocytes to restore or replace bone, cartilage, skin, and liver tissue respectively.
The present invention includes a hydrogel formed of cross-linked keratin not requiring an added binding agent. The hydrogel is believed to be bound together by reformed disulfide linkages and hydrogen bonds. A preferred use of the hydrogel is as a wound healing agent. Another preferred use is as a tissue engineering cell scaffold for implant applications. Yet another preferred use is as a skin care product. The hydrogel can be formed from a soluble protein fraction derived from hair. Keratin can be obtained from a number of sources including human or animal hair, and finger or toe nails, with one source being hair of the patient or donors.
The hydrogel can be formed by providing clean, washed, rinsed, and dried hair. The hair is partially oxidized with an oxidizing agent such as peracetic acid. The partial oxidation cleaves some disulfide linkages while leaving others intact. The cleaved bonds can form sulfonic acid residues. The partially oxidized hair can be recovered with filtration, rinsed with deionized water, dried under vacuum, and ground to a powder.
The partially oxidized powder can then have some of the remaining intact disulfide linkages cleaved with a reducing agent such as ammonium thioglycollate in ammonium hydroxide by suspending the powder in such a reducing solution. The protein suspension can be heated to about 60xc2x0 for about 4 hours and cooled to room temperature. The cleaved disulfide linkages are reduced to form cysteine groups and cysteine-thioglycollate disulfide groups, solubilizing the protein even further. The insoluble keratin fraction is preferably removed from the suspension by centrifuging the suspension and collecting the supernatant. The supernatant is preferably purified using a method such as dialysis. The supernatant can be further concentrated, in one method, by application of vacuum at ambient or sub-ambient temperatures.
The supernatant, having keratin with sulfonic acid groups, cysteine groups, and cysteine-thioglycollate disulfide groups, is now oxidized to allow formation of disulfide linkages between protein backbones. The sulfonic acid residues remain as hydrophilic sites within the protein. The hydrophilic sites bind water in the hydrogel.
The hydrogel is thus formed of pure keratin, bound together with disulfide linkages and hydrogen bonds. The hydrogel requires no binders. The keratin hydrogel provides a non-antigenic, mitogenic wound healing agent that maintains wound moisture and provides a scaffold for cell growth for tissue engineered implants. Another application for this keratin gel is as a skin care product.
Keratin has been shown to be biocompatible, non-immunogenic, not to inhibit activated T-cells and therefore not interfere with the normal cell mediated immune response, and to be mitogenic for keratinocytes, fibroblasts, and human microvascular endothelial cells. Keratin has also been shown to promote epithelialization in wound healing studies on rats and humans.